STRUCTURE OF POLYTENE CHROMOSOME BANDS AND INTERBANDS

拟议的研究建立在最近对我们的硬件和 软件允许以通用分辨率重叠的方式，3- 在这两种光下亚细胞结构的维度重建 和电子显微镜水平。最新进展使收集成为可能 活体三维时变多波长成像的研究 信息，以及随后的量化分析，以提供见解 进入染色体结构。 我们将这种方法应用于高阶染色体的分析。 利用独特的间期多线染色体的结构和功能 黑腹果蝇。我们的重点是细胞学鉴定的多烯 染色体带、带间和泡芙(转录活性， 形态上不同的染色体位置)。我们利用果蝇 变态，由昆虫激素蜕皮激素引发，作为一种 以一个特有的转录级联反应为目标。 这项建议的具体目的是： A)继续确定具体目标的三维结构 多线染色体条带、带间和膨大的电子显微镜观察 体层摄影术。 B)研究活体结构动力学(定量运动分析) 脉冲标记的多线染色体内的DNA复制位置。 C)研究蜕皮激素诱导的泡腾的体内结构动力学 形成和回归。 D)研究多丝染色体泡状物的结构动力学 相关蛋白(蜕皮激素受体和早期泡芙编码蛋白) 与蜕皮激素反应有关。 E)使用成像共振能量转移方法，确定动力学 泡芙结构中特定的蛋白质-蛋白质关联。

The proposed research builds on recent extensions to our hardware and software that allows, in a generalized resolution overlapping fashion, 3- dimensional reconstructions of subcellular structures at both the light and electron microscopic levels. Recent progress makes possible collection of in vivo 3-dimensional time dependent multiple wavelength image information, with subsequent quantitative analysis, to provide insights into chromosome structure. We apply this methodology to the analysis of higher-order chromosome structure and function using the unique interphase polytene chromosomes of Drosophila melanogaster. We focus on cytologically identified polytene chromosome bands, interbands and puffs (transcriptionally active, morphologically distinctive chromosome sites). We utilize Drosophila metamorphosis, triggered by the insect hormone ecdysone, as a way to target a characteristic transcriptional cascade. The specific aims of this proposal are to: A) Continue the determination of the 3-dimensional structure of specific polytene chromosome bands, interbands and puffs using electron microscope tomography. B) Study the in vivo structural dynamics (quantitative motion analysis) of pulse labeled DNA replication sites within polytene chromosomes. C) Study the in vivo structural dynamics of ecdysone induced puff formation and regression. D) Study the structural dynamics, on polytene chromosome puffs, of proteins (ecdysone receptor and early puff encoded proteins) associated with the ecdysone response. E) Using imaging resonance energy transfer methods, determine the dynamics of specific protein-protein associations within puff structures.